Influenza remains a worldwide public health threat in the 21st Century. Its impact is magnified by a vaccine that affords incomplete protection whose composition usually changes annually, and by the episodic appearance of new pandemic strains, such as the A/California H1N1 strain that emerged in 2009. Influenza has a particularly acute impact in in the geriatric age group, with 90% of the 20,000-40,000 annual deaths in the United States attributed to influenza occurring in individuals over the age of 65. This proposal, like all of the Projects in this Human Immunology Project Consortium application, seeks to identify the host factors associated with immune response in humans. Here we focus on factors contributing to influenza vaccine response, and how these factors and pathways are affected by age and functional status. In this proposal, we will leverage our expertise in carrying out studies in human immunology, particularly our studies that have identified innate immune and gene expression signatures of vaccine response to standard-dose vaccine and how aging affects these signatures. In particular, we found an unexpected mitochondrial biogenesis signature that was strongly associated with vaccine antibody response, and this finding, together with preliminary metabolomic data, has led us to propose employing already-collected serum samples to derive biochemical signatures of vaccine response that will be integrated with the gene expression, cellular and immunologic signatures already elucidated for young and older adults in this cohort. We also propose to elucidate new insights into the biologic basis of frailty, a geriatric syndrome characterized by decreased physiologic reserve and stress resistance that is associated with increased mortality and disability. We will also gain new insights into the high-dose influenza vaccine recently approved for older adults, administering this vaccine to a cohort of young (age 21- 30), non-frail older and frail older (? 70) adults. For these studies, we will leverage large databases of older adults established by the Yale Program on Aging and the expertise and track record of experts in frailty and disability at Yale such as Dr. Thomas Gill. Samples obtained prior to and following vaccination will undergo detailed analyses by CyTOF (Core C) for innate and adaptive immune cell composition (including novel studies of the platelet lineage), activation, and cytokine production. We will also carry out unbiased studies of innate immune pattern recognition receptor function. These findings will be integrated with single-cell RNA-seq studies utilizing the nanowell platform in Core C to isolate individual cells from lineages identified to change in the context of vaccination, and with metabolic analyses of serum and cells pre- and post-vaccine. Such metabolomic analyses will identify pathways reflecting the integration of numerous genetic signaling inputs, and will provide additional insights into human influenza vaccine response. Insights into genetic, immunologic and metabolic architecture of influenza vaccination are likely to identify pathways that could be targets of therapies, drugs or other biological treatments to enhance or suppress immune responses as needed.